The tight junctions (tj) or zonula occludens (ZO) are one of the hallmarks of absorptive and secretory epithelia (Madara, J. Clin. Invest., 83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990)). Tight junctions act as a barrier between apical and basolateral compartments, selectively regulating the passive diffusion of ions and water-soluble solutes through the paracellular (between cells) pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol. 22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond, Physiologist, 20:10-18 (1977)).
Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, since the resistances of enterocyte plasma membranes are relatively high (Madara, supra). The ZO represents the major barrier in this paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on the number of transmembrane protein strands, and their complexity in the ZO, as observed by freeze-fracture electron microscopy (Madara et al, J. Cell Biol., 101:2124-2133 (1985)).
Tight Junction Dysfunctions
Tight junction dysfunction occurs in a variety of clinical conditions, including food allergies, infections of the gastrointestinal tract, autoimmune diseases, Celiac disease and inflammatory bowel diseases (Fasano, A., Pathological and therapeutical implications of macromolecule passage through the tight junction. In Tight Junctions, CRC Press, Inc., Boca Raton, Fla. 697-722 (2001)). Healthy, mature gut mucosa with its intact tight junction serves as the main barrier to the passage of macromolecules. During the healthy state, small quantities of immunologically active antigens cross the gut host barrier. These antigens are absorbed across the mucosa through at least two pathways. Up to 90% of the absorbed proteins cross the intestinal barrier via the transcellular pathway, followed by lysosomal degradation that converts proteins into smaller, non-immunogenic peptides. These residual peptides are transported as intact proteins through the paracellular pathway, which mediates a subtle, but sophisticated, regulation of intercellular tight junction that leads to antigen tolerance.
When the integrity of the tight junction system is compromised, in premature infants or after exposure to radiation, chemotherapy, or toxins, a deleterious immune response to environmental antigens, resulting in autoimmune diseases and food allergies, may be elicited. In normal bowels, the immune reaction is regulated to maintain homeostasis of the gut.
Celiac disease (CD) is a chronic autoimmune disease that is HLA-DQ2/DQ8 haplotype restricted. Gluten, the major protein fraction of wheat, and related proteins in rye and barley are the triggering agents of the disease. Ingested gluten or its derivative fractions (gliadin and subunits) elicit a harmful T cell-mediated immune response after crossing the small bowel epithelial barrier, undergoing deamidation by tissue transglutaminase (tTG) and engaging class II MHC molecules. When the integrity of the tight junction system is compromised, as in CD, a paracellular leak (“leaky gut”) and an inappropriate immune response to environmental antigens (i.e., gluten) may develop.
Inflammatory bowel disease (IBD) is a phrase used to describe an inappropriate immune response that occurs in the bowels of affected individuals. Two major types of IBD have been described: Crohn's disease and ulcerative colitis (UC). Both forms of IBD show abnormal profiles of T cell-mediated immunity. In the gut of a person with Crohn's disease a strong Th1 reaction is induced; the Th2 response is upregulated in the colon of a UC sufferer.
The barrier function of the intestines is impaired in IBD. For example, Crohn's disease is associated with increased permeability of the intestinal barrier even in quiescent patients (Oshitani, et al., Int. J. Mol. Med. 15(3):407-10 (2005)). A TNF-α-induced increase in intestinal epithelial tj permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions (see Ye, et al., Am. J. Physiol.-Gastro. and Liver Physiol., 290(3):496-504 (2006)). Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex (Willemsen, et al., Clin. Exp. Immunol. 142(2): 275-284 (2005)).
Acute Respiratory Distress Syndrome (ARDS) presents in about 150,000 individuals in the US annually, with a mortality rate of 30-50%. ARDS occurs in response to diverse forms of severe injury, in which lung edema results in respiratory failure. The current standard of care for ARDS is limited to the management of the disease through supportive mechanical ventilation. The loss of endothelial barrier integrity is central to the pulmonary edema that occurs in ARDS.
Triggering causes for ALI (Acute Lung Injury) including ARDS can, for example, be diffuse pulmonary infections (e.g. due to viruses, bacteria, fungi), aspiration of liquids (e.g. gastric juice or water), inhalation of toxins or irritants (e.g. chlorine gas, nitrogen oxides, smoke), direct or indirect trauma (e.g. multiple fractures or pulmonary contusion), systemic reactions to inflammations outside the lung (e.g. hemorrhagic pancreatitis, gram-negative septicemia), transfusions of high blood volumes or alternatively after cardiopulmonary bypass.
The pulmonary vascular endothelium lines the intravascular space and presents a selective barrier that actively regulates paracellular movement of circulating fluid, macromolecules, and cells, into extravascular tissues and compartments. Loss of this endothelial barrier integrity is the central defect found in acute lung injury (ALI) and ARDS. The host response to a wide range of injurious stimuli includes the biosynthesis and release of endogenous mediators, some of which can open the paracellular pathway in lung microvascular endothelia. Several of these mediators have been identified, including tumor necrosis factor α, interleukin-1, thrombospondin-1, and SPARC/osteonectin, and established as factors that disrupt endothelial barrier integrity. In recent preliminary studies, we found that ΔG, the active domain of zonula occludens toxin (ZOT) of Vibrio cholerae, increases paracellular permeability across human lung microvascular endothelial cells (HMVEC-Ls).
Disruption of lung tight junction function has been implicated in the development of allergic sensitization and asthma. In order for an allergen to reach antigen-presenting cells and induce an immune response, such as in asthma, the allergen must cross the lung epithelium. It has been shown that dust mite allergen Der p 1 causes disruption of lung tight junction structure and an increase in the permeability of lung epithelia. Wan, et al. J. Clinical Investigation 104(1):123-133 (1999). It was suggested that the transepithelial movement of Der p 1 may have been facilitated by the inherent proteolytic activity of Der p 1.
Antagonism and/or inhibition of intestinal tight junction function has been demonstrated to slow, delay and/or reverse the progression toward diabetes. (see U.S. Pat. No. 7,026,294 and US Application Publication No. 20060287233). Thus, the compositions and methods of the present invention are believed to be useful to prevent and or delay the onset of diabetes, and to mitigate the long-term complications of diabetes. Further, the permeability changes associated with autoimmune diseases are long standing, and early intervention using the compositions and methods of the present invention is believed to have untold benefits to the diabetic patient.
Zonula occludens toxin (ZOT), which is produced by Vibrio cholerae, has been characterized by Fasano et al., (Proc. Natl. Acad. Sci., USA, 8:5242-5246 (1991)) and the sequence has been determined (GenBank accession no. A43864). ZOT increases the intestinal permeability of rabbit ileal mucosa by modulating the structure of intercellular tight junctions. Mammalian proteins that are immunologically and functionally related to ZOT have been identified. See U.S. Pat. No. 5,945,510. These proteins, referred to as “zonulin,” function as the physiological effector of mammalian tight junctions. These proteins are useful for enhancing absorption of therapeutic agents across tight junctions of intestinal and nasal mucosa, as well as across tight junctions of the blood brain barrier.
ZOT and ΔG have been previously identified as tight junction agonists, i.e., compounds that mediate or facilitate or augment the physiological, transient opening of tight junctions that form a barrier between adjacent epithelial cells. The ability of ZOT and ΔG to open tight junctions has been used to facilitate the transfer of macromolecule across epithelial barriers (see U.S. Pat. No. 5,665,389 and Salama et al. J. Pharmacology and Experimental Therapeutics 312(1):199-205, 2005). ZOT has been shown to act as a tight junction agonist that allows opening of tight junctions between adjacent mucosal epithelial cells. Compounds that antagonize the opening of tight junctions have been identified (see U.S. Pat. Nos. 6,458,925, 6,670,448, 6,936,689 and 7,189,696). One such antagonist, AT-1001, is currently in Phase II clinical trials for the treatment of celiac disease where it protects against loss of gut mucosal barrier function.
Peptide antagonists of tight junction opening were described in U.S. Pat. No. 6,458,925 (corresponds to WO 00/07609), which is incorporated by reference herein in its entirety. Peptide antagonists of tight junction opening may bind to the receptor utilized by the zonula occludens toxin expressed by Vibrio cholerae, and not function to physiologically modulate the opening of mammalian tight junctions. The peptide antagonists may competitively inhibit the binding of ZOT and/or zonulin to the ZOT receptor, thereby inhibiting the ability of ZOT and/or zonulin to physiologically modulate the opening of mammalian tight junctions.
There remains a need in the art for compositions and methods to treat such diseases characterized by excessive or undesirable permeability of tissues containing tight junctions. This need and others are met by the present invention.